Pest infestation, disease, and adverse environmental conditions can result in severe crop damage or loss. In the Western world, crop devastation can translate into financial ruin for those involved in the agricultural industry. In many other parts of the world the results may be even more drastic including widespread malnutrition and famine. There exists a continuing need to develop plants and crops that exhibit improved resistance to plant stresses, thereby increasing crop yields in adverse conditions and reducing the risk of crop failure. For example, plants with increased tolerance to drought, heat and higher salt conditions may open the possibility of farming in semi-desert climates, where agriculture was previously non-viable. Conversely, the development of novel crops with improved tolerance to cold or freezing temperatures may significantly prolong the growing season in regions with colder climates.
A number of plant genes are known to show increased levels of expression when plants are exposed to stress. Examples include those genes involved in metabolic pathways influenced by abscisic acid; a naturally occurring plant ‘growth hormone’ that can promote several plant functions including, for example, leaf aging, apical dominance, and seed or bud dormancy. The levels of abscisic acid are known to increase in plants under stress. Moreover, exogenous application of abscisic acid to plants is known to increase tolerance to abiotic stresses including chilling, cold, heat, salt and dehydration (Guy (1990) Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 187-223.)
Previously, the inventors for the present invention have shown that the application of 75 μM abscisic acid to cell suspension cultures of Bromus inermis can result in increased freezing tolerance, with a corresponding increase and de novo synthesis of a specific set of unknown proteins (Robertson et al. 1987 Plant Physiol. 84: 1331-1337; Robertson et al. 1988 Plant Physiol. 86: 344-347). Additional studies have shown that abscisic acid treated bromegrass cells can exhibit an increased tolerance to heat (Robertson et al. 1994 Plant Physiol. 105: 181-190), and salt (Ishikawa et al. 1995 Plant Science 107: 83-93).
Studies using comparative 2-dimensional gel electrophoresis have indicated that a large number of unknown proteins may be upregulated in response to stress (Robertson et al. 1994). Some of these proteins in the 20-30 kDa size range are cross-reactive with an anti-dehydrin antibody and an antibody (Wcs120) to cold-responsive winter wheat protein. Another group of proteins in the 43-45 kDa range were differentiated from those in the 20-30 kDa range by a lack of cross-reactivity with Wcs120 and poor cross-reactivity with the anti-dehydrin antibody. Moreover, some of the proteins in the 43-45 kDa range were found by microsequencing to have some degree of homology within the initial amino-terminal amino acids.
Despite considerable efforts to engineer genetically modified crops with increased stress tolerance, to date there are little or no such crops on the commercial market. Performance Plants Inc. have reported a drought tolerant canola plant with modified stomatal function that shows 10% increased yield over controls under drought conditions. Transgenic tomato plants (Zhang, H-X and Blumwald, E. 2001. Nature Biotech, 19: 765-768) with enhanced salinity were produced by overexpressing a vacuolar Na+/H+ antiport protein. The freezing tolerance of non-acclimated and cold acclimated canola seedlings can be increased by over expressing CBF (C-repeat/dehydration responsive element binding factor) (Jaglo et al. 2001. Plant Physiol, 103(4): 1047-1053). This work was based on the observation that small increases in freezing tolerance occurred in Arabidopsis seedlings constitutively expressing CBF genes (Gilmour, S. J. et al. 1998. Plant J., 16: 433-442.) Enhanced tolerance to both salt and drought stresses has been identified in transgenic Arabidopsis plants overexpressing vacuolar H+-pyrophosphatase (Gaxiola, R. A. et al. 2001. Proc. Natl. Acad. Sci. USA, 25: 11444-11449). Most transgenic plant work in abiotic stress has been done with Arabidopsis thaliana a non-economic model plant system.
The future prospects of engineering novel plants with an increased capacity to tolerate environmental insults will depend on the availability of critical stress tolerance controlling genes, and knowledge of their functional regulatory properties; The inventors for the present application, and others, have endeavored to decipher the mechanisms of plant stress tolerance in the hope of developing an understanding of the biochemical pathways involved. Nonetheless, the characterization of the genes and proteins involved in plant stress responses presents a number of significant challenges.
There remains a continuing need to develop a better understanding of plant stress responses, so that corresponding methods can be developed to confer advantageous properties to plants. This need extends to the production of crops with an increased capacity to resist damage by both infestation and disease. In addition, there remains a need to develop crops that exhibit resistance to damage by adverse climatic conditions such as excessive temperatures, drought, flood, low levels of nutrients, or high levels of toxins. Even incremental gains in plant stress tolerance may have a significant economic impact in stablizing the quality and supply of grain, oilseed and horticulture. Enhancement of germination, growth and flowering are extremely important in regions that have a short or otherwise difficult growing season.